Jennie Hillmann

A Review of "A Novel Family of Mammalian Taste Receptors" by Adler, Hoon, Mueller, Chandrashekar, Ryba, and Zuker, 2000

G-protein-coupled receptors (GPCRs) are thought to mediate the signaling process in taste transduction of sweet, bitter, and umami taste. The way in which the signal transduction is translated into taste perception, however, remains unclear. Olfactory perception is process that is more clearly understood than taste perception. Research in mammal olfactory systems indicates that single olfactory receptors recognize multiple odorants and that a single odorant is recognized by multiple receptors. This overlap in receptor recognition demonstrates that perception of odors involves combining these signals into a code that allows for very accurate discrimination of odors. Taste encoding could be similar to mammalian olfactory perception, or it could be more like the worm olfactory system, which can recognize a wide diversity of odors with its hundreds of olfactory receptors, but processes the odors along only a few chemosensory neurons. Worm olfactory perception, then, allows for recognition of diverse odorants, but is limited in its ability to discriminate between the odorants. Research in taste perception attempts to identify the receptors involved in taste coding, to determine the function of individual receptors, and to elucidate the information coding process. This paper by Adler et al. claims to identify a new family of taste receptors called T2Rs and to demonstrate that the structure and location of the T2R implicate the receptor in bitter taste recognition.
Gustducin is a G-protein alpha subunit expressed in taste cells. Because T1Rs (another family of GPCRs) are not coexpressed with gustducin in in situ hybridization studies, Adler et al. assumed that there must be another family of GPCRs that are expressed in gustducin-positive cells. Because gustducin has been linked to bitter taste perception, the authors searched DNA databases for GPCRs located in genomic intervals associated with a locus for PROP, which is thought to mediate the response to the bitter substance 6-n-propyl-2-thiouracil. An analysis of open reading frames in a 450 kb section of DNA from six different human genomic BAC clones identified the T2R-1. The authors then performed computer searches using the T2R-1 and found 19 additional human receptors.
Figure 1 shows alignment of predicted amino acid sequences for human, rat, and mouse T2Rs. Interestingly, the sequence alignment shows the highest conservation in the seven transmembrane segments, and vast diversity in the extracellular portions of the protein, which they suggests indicates the need to recognize a diverse group of ligands. Figure 2 shows a cladogram demonstrating the relationships between different full-length rat, mouse, and human T2Rs, as well as the relationships of these T2Rs to vomeronasal receptors and opsin, proteins to which T2Rs are distantly related.
Figure 3 maps genes to their loci on chromosomes and demonstrates that they are in the correct loci to be involved in bitter taste reception. The figure shows a map of human chromosomes 5, 12, and 7 and mouse chromosomes 15 and 6. Homologous regions between mouse chromosome 6 and human chromosome 7, mouse chromosome 6 and human chromosome 12, and mouse chromosome 15 and human chromosome 5 are indicated. T2Rs are located within each of these homologous regions. The T2Rs that map to homologous chromosomes turn out to be the pairs of T2Rs shown to be most closely related in the cladogram. Two of the three human T2Rs that map to homologous regions are located in the same locations as PROP. The T2R clusters together on the chromosome as head to tail arrays. Clusters of T2Rs are shown from human chromosomes 12 and 7 as well as mouse chromosome 6. Arrowheads within the 9 gene cluster and the 4 gene cluster from human chromosomes 12 and 7 indicate the direction of transcription. All of the T2Rs seem to be transcribed in the same direction, while PRP genes are transcribed in the opposite direction. PRP, presumably, is another gene involved in bitter taste perception, but the authors never explicitly state the role of PRP. The 9 gene cluster also maps to a region that is homologous with the bitter cluster containing region on mouse chromosome 6, where the remaining 25 mouse T2Rs are located. The locus of the PROP gene maps to the T2R containing regions of human chromosomes 7 and 5. Interestingly, an 18 base pair sequence was found located in the 5' upstream sequences of the majority the T2Rs. This sequence is quasi-palindromic (its complementary sequence is almost exactly the sequence read backwards). There were a few of these sequences found that were not just upstream of a T2R suggesting that there are more T2Rs that are not mapped. (They estimate that there are 80 to 120 T2Rs.) The order of some of the mouse T2Rs are not known, and, therefore, the three BAC (Bacterial Artificial Chromosome) contigs may not be shown in an accurate orientation.
The taste bud containing regions of the rodent head are shown in Figure 4. They include the fungiform papillae, the foliate papillae, the circumvallate papilla, the epiglottis, and the geschmackstreifen. If the T2R is in fact a functional taste receptor, it should be located in some or all of the taste bud containing regions. To determine whether or not T2Rs are located in these regions, the authors performed in situ hybridization rat of circumvallate papilla taste buds probing with antisense cRNA probes of five different T2Rs. Probes bound to an average of two cells per taste bud, which is approximately 15% of the cells in a taste bud. Figure 5a-5e demonstrate the hybridization of each of the five T2Rs. Though the data is not shown, the authors say they have seen similar results with an additional 11 rat T2Rs and with mouse sections hybridized with 17 mouse T2R probes. Figure 5f, g, and h show the hybridization of the probes to cells in the foliate papillae, geschmackstreifen, and epiglottis respectively. The data only show the hybridization of one probe to each of these. Also, the images in 5g and h are limited to two and one taste buds, whereas the other images show many taste buds. This is probably because taste buds are more spread out in these other areas, but it is not clear from the information given. When they probed hundreds of fungiform receptors with 11 different T2R probes the authors found that less than 10% of fungiform papillae contain cells with T2Rs. The few fungiform cells that do express T2Rs, however, contain multiple positive cells (Figure 5i).
Adler et al. reasons that if about 15% of the cells in the circumvallate, foliate, and palate taste buds express T2Rs and there are over 30 T2Rs in the rodent genome, then a single taste cell must express more than one type of T2R. Figure 6a-c demonstrates the results of in situ hybridization with mixes of 2, 5, and 10 T2Rs in the various taste bud containing areas. Even with mixures of 10 different T2Rs, the probes hybridized to only a few more cells (5% more) than an individual T2R probe, though the signal intensity is increased when multiple probes are used. Though the data are not shown, the authors report similar results for taste bud containing regions including the fungiform papilla. When double label hybridization was performed using fluorescein or digoxigenin, both labels hybridized in the same regions of the same taste buds, indicating that the cells are expressing more than one type of receptor (Figure 6d).
Finally Adler et al. wants to demonstrate that T2Rs are expressed in gustducin-expressing cells (an important demonstration if they initially searched for another type of
GPCR for the reason that T1Rs were not coexpressed in gustducin-expressing cells, and another GPCR, then, must be involved). They perfomed in situ hybridizations using diferentially labeled gustducin and T2Rs. The results in Figure 7a and b do seem to show a strong overlap of cells expressing T2Rs and cells expressing gustducin. They say that approximately a third of all gustducin producing cells are also T2R-expressing cells in the circumvallate, foliate, and palate taste buds. The authors also say that very few cells in the front of the tongue that are gustducin-expressing are also T2R-expressing. To be sure that T2R were not simply being expressed at a level below their detection limits they performed PCR amplification using T2R specific primers, the results of which still showed no more T2R-expressing cells. They also believe it to be unlikely that they missed the detection of additional T2R-expressing cells because they probed with several different T2Rs, and because the few cells that did express T2R showed hybridization of all of the T2R probes. The authors take this as evidence that another set of GPCRs are functioning in gustducin-expressing cells as well.
Additional results are reported, but the data is not shown for evidence of gustducin-expressing cells in certain regions of the gastrointestinal tract, trachea, pharynx, nasal respiratory epithelium, ducts of salivary glands, and vomeronasal organ, some of which are positive for T2R expression. Adler et al. says that these results suggest a possible role for these cells in chemoreception and strengthen the argument for T2Rs as gustducin-linked receptors.
The evidence provided clearly demonstrates the identification of new receptor that is localized to all of the taste bud containing areas. The results also strongly suggest that the T2R is closely linked to gustducin expression. It even seems as though T2Rs are selectively expressed in cells that are gustducin-expressing. The authors indicate that T2R's involvement in bitter taste reception is very likely, but they have not yet even demonstrated that T2R in fact functions as a taste receptor. While the robust nature of the results for the in situ hybridization experiments provides a convincing story for the overlap of labelling for different T2R probes as well as the overlap for labelling of T2Rs and gustducin, the figures that fluorescently label each protein separately (Figures 6d, and 7a and b) should have shown controls with which to compare them. The methods section reports that they performed controls using sense probes, which should have given no significant signal (negative control) and with G alpha i subunit cDNA which should have produced a signal in every taste cell (positive control), and found the expected results. Even in the absence of these controls for comparison, the data are still convincing. The authors hypothesize that bitter taste perception is more akin to the worm olfactory system, which is limited in its discriminatory power, than the mammalian olfactory system, given the existence of complex repertoire T2Rs, the demonstration that multiple T2Rs are expressed in each taste cell, and the fact that humans perceive bitter tastes in a uniform manner.
Cleary the next steps in research with the T2R family need to involve testing the function of the T2R as a taste receptor, and then as a bitter taste receptor. This will require finding a way to demonstrate the activation of the T2R in response to bitter tastants. The protein should also be characterized. Adler et al. shows the transmembrane domains of the T2R in Figure 1, but never gives a predicted characterization of the shape of the protein. As the authors indicate, there is most likely one or more additional receptor types involved in bitter taste perception. Finding additional receptor types for bitter taster perception would involve finding a locus involved in responding to a bitter substance other than PROP and searching for DNA sequences near this alternate locus.
A follow-up paper from the same lab by Chandrashekar et al. actually performs functional tests to determine T2Rs role as a bitter taste receptor. They target T2Rs to the plasma membrane by making them into chimeric proteins with rhodopsin sequences. Rhodopsin is responsible for relocating the protein to the plasma membrane. The T2Rs are also coupled to G-alpha-15, a subunit that allows for single cell detection of a calcium response following activation of the protein. A calcium response is detected only when the T2Rs are activated with bitter tastant cycloheximide, denatonium, or PROP. The activation responses are shown to be specific to bitter tastants, and also show desensitization to repeated stimulation with bitter tastants. When Chandrashekar et al. tested stimulation with a variety of bitter and sweet tastants, they found that T2Rs are limited to certain bitter tastants, and are not activated by sweet tastants. In particular, human T2Rs responded only to denatonium and PROP. Mouse strains shown be deficient in their ability to detect cycloheximide (the bitter tastant specific for the mouse T2R) show amino acid substitutions. T2Rs from this mouse strain show a significantly decreased response to cycloheximide stimulation relative to wild type strains. In demonstrating the amino acid substitution, the authors give a predicted membrane topology characterizing the protein. Importantly, the authors demonstrate the sufficiency of the T2R to for bitter taste perception of specific bitter tastants. They expressed a mouse T2R in insects and then activated the receptor with cycloheximide. Cycloheximide stimulation showed gustducin activation, indicating that T2R is coupled to gustducin, and offering further support of the role of T2R in bitter taste perception.
In order to understand how bitter tastes are encoded and to determine if bitter taste perception is, in fact, uniform despite the diversity of T2Rs, it would be interesting to perform electrophysiological analyses of the neural responses to stimulation of the T2R. If the physiological response of different T2Rs is uniform, then it would indicate that perception of bitter taste is uniform. However, if the physiologcal response of different T2Rs is varied, it would indicate that there may subtle discriminatory power in bitter taste perception. If more T2R are discovered (possibly by taking advantage of the 18 kb quasi-palindromic sequence as an indicator of a T2R downstream), then functional tests of the full repertoire of T2Rs might reveal that T2Rs can respond to more than just the specific bitter tastants highlighted in the Chandrashekar et al. paper.

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